This invention relates to polypeptide hormone analogues that exhibits enhanced pharmaceutical properties, such as increased biological potency, more rapid pharmacokinetics, and/or augmented resistance to thermal fibrillation above room temperature. More particularly, this invention relates to insulin analogues that are modified by attachment of O-link carbohydrate moieties at positions B27 and/or B30. Such non-standard sequences may optionally contain standard amino-acid substitutions at other sites in the A or B chains of an insulin analogue.
The engineering of non-standard proteins, including therapeutic agents and vaccines, may have broad medical and societal benefits. An example of a medical benefit would be optimization of the pharmacokinetic properties of a protein. An example of a further societal benefit would be the engineering of proteins more refractory than standard proteins with respect to degradation at or above room temperature for use in regions of the developing world where electricity and refrigeration are not consistently available. An example of a therapeutic protein is provided by insulin. Analogues of insulin containing non-standard amino-acid substitutions may in principle exhibit superior properties with respect to biological activity, pharmacokinetics or resistance to thermal degradation. The challenge posed by the pharmacokinetics of insulin absorption following subcutaneous injection affects the ability of patients to achieve tight glycemic control and constrains the safety and performance of insulin pumps. The challenge posed by its physical degradation is deepened by the pending epidemic of diabetes mellitus in Africa and Asia. These issues are often coupled as modifications known in the art to accelerate absorption following subcutaneous injection usually worsen the resistance of insulin to chemical and/or physical degradation. Because fibrillation poses the major route of degradation above room temperature, the design of fibrillation-resistant formulations may enhance the safety and efficacy of insulin replacement therapy in such challenged regions. The present invention pertains to the use of an O-linked carbohydrate-modified residue at B27 and/or B30 in combination with standard or non-standard substitutions elsewhere in the A-chain or B-chain or in combination with C-terminal extension of the B-chain as a novel strategy to modify and improve distinct properties of insulin. During the past decade specific chemical modifications to the insulin molecule have been described that selectively modify one or another particular property of the protein to facilitate an application of interest. Whereas at the beginning of the recombinant DNA era (1980) wild-type human insulin was envisaged as being optimal for use in diverse therapeutic contexts, the broad clinical use of insulin analogues in the past decade suggests that a suite of analogues, each tailored to address a specific unmet clinical need, would provide significant medical and societal benefits.
Administration of insulin has long been established as a treatment for diabetes mellitus. Insulin is a small globular protein that plays a central role in metabolism in vertebrates. Insulin contains two chains, an A chain, containing 21 residues, and a B chain containing 30 residues. The hormone is stored in the pancreatic β-cell as a Zn2+-stabilized hexamer, but functions as a Zn2+-free monomer in the bloodstream. Insulin is the product of a single-chain precursor, proinsulin, in which a connecting region (35 residues) links the C-terminal residue of B chain (residue B30) to the N-terminal residue of the A chain (FIG. 1A). A variety of evidence indicates that it consists of an insulin-like core and disordered connecting peptide (FIG. 1B). Formation of three specific disulfide bridges (A6-A11, A7-B7, and A20-B19; FIGS. 1A and 1B) is thought to be coupled to oxidative folding of proinsulin in the rough endoplasmic reticulum (ER). Proinsulin assembles to form soluble Zn2+-coordinated hexamers shortly after export from ER to the Golgi apparatus. Endoproteolytic digestion and conversion to insulin occurs in immature secretory granules followed by morphological condensation. Crystalline arrays of zinc insulin hexamers within mature storage granules have been visualized by electron microscopy (EM). The sequence of insulin is shown in schematic form in FIG. 1C. Individual residues are indicated by the identity of the amino acid (typically using a standard three-letter code), the chain and sequence position (typically as a superscript). Pertinent to the present invention is the designation of individual atoms in the amino acids Serine and Threonine, which each contain a hydroxyl group (—OH) attached to the beta carbon (Cβ) of their respective side chains. By convention the oxygen atom attached to Cβ is denoted Oβ.
The pharmacokinetic features of insulin absorption after subcutaneous injection have been found to correlate with the rate of disassembly of the insulin hexamer. Although not wishing the present invention to be constrained by theory, modifications to the insulin molecule that lead to accelerated disassembly of the insulin hexamer are thought to promote more rapid absorption of insulin monomers and dimers from the subcutaneous depot into the bloodstream. A major goal of insulin replacement therapy in patients with diabetes mellitus is tight control of the blood glucose concentration to prevent its excursion above or below the normal range characteristic of healthy human subjects. Excursions below the normal range are associated with immediate adrenergic or neuroglycopenic symptoms, which in severe episodes lead to convulsions, coma, and death. Excursions above the normal range are associated with increased long-term risk of microvascular disease, including retinapathy, blindness, and renal failure. Because the pharmacokinetics of absorption of wild-type human insulin following subcutaneous injection is often too slow and too prolonged relative to the physiological requirements of post-prandial metabolic homeostasis, considerable efforts have been expended during the past 20 years to develop insulin analogues that exhibit more rapid absorption with pharmacodynamic effects that are more rapid in onset and less prolonged in duration. Examples of such rapid-acting analogues known in the art are [LysB28, ProB29]-insulin (KP-insulin, the active component of Humalog®), [AspB28]-insulin (Novalog®), and [LysB3, GluB29]-insulin (Apidra®). These products contain standard amino-acid substitutions, which change the pattern of positive or negative charges in the molecule and which, in the case of KP-insulin or [AspB28]-insulin, respectively relocate or remove the distinctive pyrrolidine ring of Proline (an imino acid) at position B28. Although widely used in clinical practice, these analogues exhibit two principal limitations. First, although their pharmacokinetic and pharmacodynamic profiles are more rapid than those of wild-type insulin, they are not rapid enough in many patients to optimize glycemic control or enable the safe and effective use of algorithm-based insulin pumps (closed-loop systems). Second, the amino-acid substitutions in these analogues impair the thermodynamic stability of insulin and exacerbate its susceptibility to fibrillation above room temperature. Thus, the safety, efficacy, and real-world convenience of these products have been limited by a trade-off between accelerated absorption and accelerated degradation. Because of this trade-off (which pertains to present products Humalog®, Novalog®, and Apidra® and which is reflected in instructions to patients in package inserts to inspect vials for signs of degradation), it has seemed not possible to identify substitutions that simultaneously enhance the rate of hexamer disassembly and retard formation of fibrils above room temperature.
Fibrillation, which is a serious concern in the manufacture, storage and use of insulin and insulin analogues for the treatment of diabetes mellitus, is enhanced with higher temperature, lower pH, agitation, or the presence of urea, guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drug regulations demand that insulin be discarded if fibrillation occurs at a level of one percent or more. Because fibrillation is enhanced at higher temperatures, patients with diabetes mellitus optimally must keep insulin refrigerated prior to use. Fibrillation of insulin or an insulin analogue can be a particular concern for such patients utilizing an external insulin pump, in which small amounts of insulin or insulin analogue are injected into the patient's body at regular intervals. In such a usage, the insulin or insulin analogue is not kept refrigerated within the pump apparatus, and fibrillation of insulin can result in blockage of the catheter used to inject insulin or insulin analogue into the body, potentially resulting in unpredictable fluctuations in blood glucose levels or even dangerous hyperglycemia. At least one recent report has indicated that insulin Lispro (KP-insulin, an analogue in which residues B28 and B29 are interchanged relative to their positions in wild-type human insulin; trade name Humalog®) may be particularly susceptible to fibrillation and resulting obstruction of insulin pump catheters. Insulin exhibits an increase in degradation rate of 10-fold or more for each 10° C. increment in temperature above 25° C.; accordingly, guidelines call for storage at temperatures <30° C. and preferably with refrigeration. The propensity of current products Humalog®, Novalog®, and Apidra® to form fibrils at or above room temperature is exacerbated on dilution, as may be used in the treatment of Type I diabetes mellitus in children or adults with low body mass. Accordingly, the shelf life of such diluted pharmaceutical formulations in use at room temperature is reduced in accordance with instructions contained in package inserts regarding disposal of diluted insulin analogue formulations. Fibrillation of basal insulin analogues formulated as soluble solutions at pH less than 5 (such as Lantus® (Sanofi-Aventis), which contains an unbuffered solution of insulin glargine and zinc ions at pH 4.0) also can limit their self lives due to physical degradation at or above room temperature; the acidic conditions employed in such formulations impairs insulin self-assembly and weakens the binding of zinc ions, reducing the extent to which the insulin analogues can be protected by sequestration within zinc-protein assemblies.
The present theory of protein fibrillation posits that the mechanism of fibrillation proceeds via a partially folded intermediate state, which in turn aggregates to form an amyloidogenic nucleus. In this theory, it is possible that amino-acid substitutions that stabilize the native state may or may not stabilize the partially folded intermediate state and may or may not increase (or decrease) the free-energy barrier between the native state and the intermediate state. Therefore, the current theory indicates that the tendency of a given amino-acid substitution in the insulin molecule to increase or decrease the risk of fibrillation is highly unpredictable. Protein- and peptide fibrils ordinarily derive from non-glycosylated proteins. Insulin, for example, contains no O-linked carbohydrate moieties (despite having Threonine residues as potential sites of modification at positions A8, B27, and B30 and having Serine residues as potential sites of modification at positions A9 and A12) and similarly no N-linked carbohydrate moieties (despite having Asparagine residues as potential sites of modification at positions A18, A21, and B3, having Glutamine at position A5, A15 and B4, and having Arginine at position B22). Although the native amino-acid sequences surrounding these residues do not specify their glycosylation in the endoplasmic reticulum or Golgi apparatus of pancreatic beta cells, such modifications may be introduced in vitro through chemical synthesis. O-linked or N-linked carbohydrate moieties at one or more of these positions would introduce a cyclic polar adduct (pyranosides) containing multiple hydrogen-bond donors and hydrogen-bond receptors. Such a residue-specific modification would therefore be expected to modify the solvation of the modified polypeptide chain and could thereby modify the relative stabilities of the native state, partially folded state, and non-native aggregates pertinent to the mechanism of fibrillation. While not wishing to be constrained by theory, it is possible that such modified glycopeptides-water interactions and potential glycopeptides-glycopeptide could augment the kinetic barrier that ordinarily protects the native state of a protein from formation of an amyloidogenic seed and could thereby delay the onset of fibrillation. While further not wishing to be constrained by theory, it seemed possible that reorganization of the water structure surrounding the carbohydrate moiety and in particular disruption of bridging water molecules involved in a network of hydrogen bonds at the outer surface of the insulin hexamer could lead to accelerated disassembly of the hexamer, thus circumventing the trade-off that thwarted previous attempts to optimize simultaneously resistance to insulin fibrillation and rate of hexamer disassembly. We further envisaged that the protective effects of carbohydrate modification would be operative under a broad range of pH conditions, including under acidic conditions employed in the formulation of basal insulin analogues (pH 3-4) whose isoelectric points are shifted to near neutrality as exemplified by insulin glargine, so long as the specific monosaccaride pyranoside adduct either did not contain a formal charge or was incorporated into a protein framework in which the overall isoelectric point was maintained to be near neutrality (pH 7) due to amino-acid substitutions or extensions elsewhere in the A- or B-chains as known in the art.
Two general considerations motivated the specific molecular designs disclosed in this application: (i) avoidance of competing high-affinity binding to membrane carbohydrate-binding proteins and (ii) avoidance of steric occlusion of the receptor-binding surface of insulin, i.e., that surface of insulin that mediates its binding to and activation of the insulin receptor. We describe these avoidance strategies as follows.                (i) Mammalian cells contain cell-surface proteins that recognize with high affinity branched-chain oligosaccharide moieties covalently tethered to proteins by O-ester linkages (to the β-hydroxyl function of Serine or Threonine) or by N-ester linkages (to the side-chain carboxamide function of Asparagine or Glutamine and to the guanadinium group of Arginine). The affinities and specificities of such membrane proteins vary but in general the strength of binding is markedly enhanced by having two or more sites of carbohydrate modification, by having branched carbohydrate moieties, and by exhibiting appropriate terminal carbohydrate units that are complementary to the binding site or binding sites of the membrane protein. High affinity is a consequence of multidentate interactions with such branching carbohydrate adducts. An example of such a membrane protein is the human mannose receptor, which binds with nanomolar or subnanomolar affinity branched-chain carbohydrate moieties that terminate in the hexose mannose or that terminate in a sulfated form of the hexose N-acetyl-galactose. Such high-affinity binding would be undesirable in an insulin analogue as binding of the hormone to a carbohydrate-binding membrane receptor would be likely to compete with binding of the hormone to the insulin receptor, which mediates the biological activities of insulin. We therefore sought to invent analogues of insulin containing single hexose modifications, intended to confer protection from fibrillation while avoiding competitive binding to and clearance by membrane carbohydrate-binding proteins. Examples of single hexose modifications are provided by an alpha-O-ester linkage between mannose, glucose, or N-acetyl-galactose to Threonine as corresponding pyranosides. The structure of mannose as a free cyclic structure (D-mannopyranoside) is shown in FIG. 2A. The structure of an N-acetyl-β-D-galactopyranoside is shown in FIG. 2B with an Oβ-linkage to Serine (right) and potentially extended by other linked carbohydrates (wavy line at left; in a monosaccaride adduct the moiety at left would terminate as —OH).        (ii) The primary receptor-binding surface of insulin spans residues A1-A14 and B6-B26. Restriction of carbohydrate modification to sites outside of these peptide segments would therefore be expected to be associated with at least a portion of the receptor-binding activity and biological potency of insulin. Examples of sites outside of the receptor-binding surface of insulin are provided by ThrB27 and ThrB30.        
Although residues B26-B30 may be deleted from insulin without loss of receptor-binding activity and so might be modifiable with retention of at least a portion of the biological potency of insulin, modifications in this segment that are known in the art to confer more rapid absorption of insulin from a subcutaneous depot also confer inferior physical stability relative to human insulin. Examples of provided by prandial insulin analogues insulin lispro ([LysB28, ProB29]-insulin, herein abbreviated KP-insulin; the active component of Humalog® (Eli Lilly and Co.)), insulin aspart (AspB28-insulin; the active component of Novolog® (Novo-Nordisk LLC)). Such impaired physical stability reflects the same structural mechanism as accelerated absorption: partial structural perturbation of the native dimerization surface of insulin (as found at three subunit interfaces of the insulin hexamer) both leads to accelerated absorption and accelerated fibrillation. It is not obvious how this segment might be modified in such a way as to confer accelerated hexamer disassembly while also protecting the insulin monomer from fibrillation. Simultaneous enhancement of the rate of hexamer disassembly and resistance to physical degradation would require a physical principle unrelated to those that underlie the design of insulin analogues that are known in the art. While not wishing to be constrained by theory, we envisaged that residue-specific modification of the solvation properties at the surface of the insulin monomer, dimer, and hexamer by means of carbohydrate modification at sites peripheral to the receptor-binding surface could simultaneously confer accelerated hexamer disassembly and enhanced protection from fibrillation with maintenance of at least a portion of the biological activity of wild-type insulin.
There is a need, therefore for an insulin analogue that displays increased resistance to fibrillation above room temperature while retaining rapid hexamer disassembly and while exhibiting at least a portion of the activity of the corresponding wild-type insulin and maintaining at least a portion of its chemical and/or physical stability.